2.1. ENDOTHELIAL CELL BIOLOGY AND BLOOD VESSEL DEVELOPMENT
The endothelium occupies a pivotal position at the interface between the circulating humoral and cellular elements of the blood, and the solid tissues which constitute the various organs. In this unique position, endothelial cells regulate a large number of critical processes. Such processes include leukocyte adherence and transit through the blood vessel wall, local control of blood vessel tone, modulation of the immune response, the balance between thrombosis and thrombolysis, and new blood vessel development (Bevilacqua et al., 1993, J. Clin. Invest 91:379-387; Folkman et al., 1987, Science 235:442-447; Folkman et al., 1992, J. Biol. Chem. 267:10931-10934; Gimbrone, 1986, Churchill Livingstone, London; Issekutz, 1992, Curr. Opin. Immunol. 4:287-293; Janssens et al., 1992, J. Bio. Chem. 267:14519-522; Lamas et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:6348-6352; Luscher et al., 1992, Hypertension 19:117-130; Williams et al., 1992, Am. Rev. Respir. Dis. 146:S45-S50; Yanagisawa, et al., 1988, Nature 332:411-415).
Endothelial cell dysfunction has been postulated as a central feature of vascular diseases such as hypertension and atherosclerosis. In this context, the ability of the endothelium to synthesize smooth muscle cell mitogens and factors which control smooth muscle contraction has received much attention (Janssens et al., 1992, J. Biol. Chem. 267:14519-14522; Lamas et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:6348-6352; Luscher et al., 1992, Hypertension 19:117-130; Raines et al., 1993, Br. Heart J. 69:S30-S37; Yanagisawa et al., 1988, Nature 332:411-415). The endothelial cell has also become the focus of attention in the study of diseases which are not primarily vascular in nature. Diverse disease processes such as adult respiratory distress syndrome, septic shock, solid tumor formation, tumor cell metastasis, rheumatoid arthritis, and transplant rejection are now understood to be related to normal or aberrant function of the endothelial cell. A rapidly increasing number of pharmacologic agents are being developed whose primary therapeutic action will be to alter endothelial cell function. In addition, recent attention on gene therapy has focused on the endothelial cell (Nabel et al., 1991, J. Am. Coll. Cardiol. 17:189B-194B). Transfer of genes into the endothelial cell may afford a therapeutic strategy for vascular disease, or the endothelium may serve simply as a convenient cellular factory for a missing blood borne factor. Hence, information regarding fundamental processes in the endothelial cell will aid the understanding of disease processes and allow more effective therapeutic strategies.
Studies from a number of laboratories have characterized the ability of the endothelial cell to dramatically alter basic activities in response to cytokines such as tumor necrosis factor (TNF)-alpha. TNF-alpha stimulation induces significant alterations in the production of vasoactive compounds such as nitric oxide and endothelin, increases surface stickiness toward various types of leukocytes, and modulates the expression of both pro- and anti-coagulant factors (Cotran et al., 1990, J. Am. Soc. Nephrol. 1:225-235; Mantovani et al., 1992, FASEB J. 6:2591-2599). In turn, endothelial cells have been shown to be an important source for the production of cytokines and hormones, including interleukin 1, 6 and 8 (Gimbrone et al., 1989, Science 246:1601-1603; Locksley et al. 1987, J. Immunol. 139:1891-1895; Loppnow et al., 1989, Lymphokine. Res. 8:293-299; Warner et al., 1987, J. Immunol. 139:1911-1917).
The ability of endothelial cells to produce granulocyte, granulocyte-macrophage, and macrophage colony stimulating factors has led to speculation that endothelial cells are an important facet of hematopoietic development (Broudy et al., 1987, J. Immunol. 139:464-468; Seelentag et al., 1987, EMBO J. 6:2261-2265). Early studies have provided the foundation for the cloning of a large number of "endothelial cell-specific" genes. Some of these include ICAM-1, ICAM-2, VCAM1, ELAM-1, endothelin-1, constitutive endothelial cell nitric oxide synthetase, thrombomodulin, and the thrombin receptor (Bevilacqua et al., 1989, Science 243:1160-1165; Jackman et al., 1986, Proc. Natl. Acad. Sci. U.S.A. 83:8834-8838; Janssens et al., 1992, J. Bio. Chem. 267:14519-14522; Lamas et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:6348-6352; Osborn et al., 1989, Cell 59:1203-1211; Staunton et al., 1989, Nature 339:61-64; Staunton et al., 1988, Cell 52:925-933; Vu et al, 1991, Cell 64:1057-1068; Yanagisawa et al., 1988, Nature 332:411-415).
All blood vessels begin their existence as a capillary, composed of only endothelial cells. Much of the molecular research investigating the role of endothelial cells in blood vessel development has focused on this process in the adult organism, in association with pathological conditions. In these situations, new blood vessels are formed by budding and branching of existing vessels. This process, which depends on endothelial cell division, has been termed angiogenesis. Research on this process has focused primarily on small proteins which are growth factors for endothelial cells (Folkman et al., 1987, Science 235:442-447; Folkman et al., 1992, J. Bio. Chem. 267:10931-10934). Sensitive bioassays for angiogenesis have allowed the characterization of a number of angiogenic factors, from both diseased and normal tissues. Members of the fibroblast growth factor (FGF) family, platelet-derived endothelial cell growth factor, and vascular endothelial cell growth factor (vascular permeability factor), are a few of the angiogenic factors which have been characterized (Folkman et al., 1987, Science 235:442-447; Folkman et al., 1992, J. Bio. Chem. 267:10931-10934; Ishikawa et al., 1989, Nature 338:557-562; Keck et al., 1989, Science 246:1309-1312; Leung et al., 1989, Science 246:1306-1309).
Such information has provided some insight into the study of blood vessel development in the embryo. Studies linking vascular development to an angiogenic factor have resulted in the work with vascular endothelial cell growth factor (VEGF). VEGF expression has been correlated in a temporal and spatial fashion with blood vessel development in the embryo (Breier et al., 1992, Development 114:521-532). A high affinity VEGF receptor, flk-1, has been shown to be expressed on the earliest endothelial cells in a parallel fashion (Millauer et al., 1993, Cell 72:835-846).
Blood vessels form by a combination of two primary processes. Some blood vessel growth depends on angiogenesis, in a process very similar to that associated with pathological conditions in the adult. For instance, the central nervous system depends solely on angiogenesis for development of its vascular supply (Noden, 1989, Am. Rev. Respir. Dis. 140:1097-1103; Risau et al., 1988, EMBO J. 7:959-962). A second process, vasculogenesis, depends on the incorporation of migratory individual endothelial cells (angioblasts) into the developing blood vessel. These angioblasts appear to be components of almost all mesoderm, and are able to migrate in an invasive fashion throughout the embryo (Coffin et al., 1991, Anat. Rec. 231:383-395; Noden, 1989, Am. Rev. Respir. Dis. 140:1097-1103; Noden, 1991, Development 111:867-876). The precise origin of this cell, and the characteristics of its differentiation have not been defined.
Understanding of the molecular basis of endothelial cell differentiation in blood vessel development may allow manipulation of blood vessel growth for therapeutic benefit. The ability to suppress blood vessel growth may also provide therapeutic strategies for diseases such as solid tumors and diabetic retinopathy. On the other hand, diseases such as coronary artery disease may be treated through pharmacologic induction of directed blood vessel growth, through increasing collateral circulation in the coronary vascular bed. Both vascular diseases such as atherosclerosis and hypertension and nonvascular diseases which depend on the endothelial cell will benefit from a better understanding of endothelial cells.
2.2. EPIDERMAL GROWTH FACTOR-LIKE DOMAIN
Epidermal growth factor (EGF) stimulates growth of a variety of cell types. EGF-like domains have been found in a large number of extracellular and membrane bound proteins (Anderson, 1990, Experientia 46(1):2; and Doolittle, 1985, TIBS, June:233). These proteins include molecules that function as soluble secreted proteins, growth factors, transmembrane signal and receptor molecules, and components of the extracellular matrix (Lawler and Hynes, 1986, J. Cell. Biol. 103:1635; Durkin et al., 1988, J. Cell Biol. 107:2749; Wu et al., 1990, Gene 86:275; Bisgrove and Raff, 1993, Develop. Biol. 157:526;).
In many cases, multiple tandem repeats of a characteristic 40 amino acid long, 6 cysteine-containing sequence are observed (Anderson, 1990, Experientia 46(1):2). EGF-like domains are homologous to the peptide growth factor EGF which consists of a single copy of the standard EGF domain. These domains have been highly conserved in evolution, being found in species as diverse as nematodes, Drosophila, sea urchins, and vertebrates.
The EGF molecule and the closely related transforming growth factor (TGF) alpha induce cell proliferation by binding to a tyrosine kinase receptor. It has been suggested that other EGF-like domains also function as ligands for receptor molecules (Engel, 1989, FEBS Lett. 251:1-7). Fundamentally, EGF repeats are protein structures that participate in specific protein-protein binding interactions.
The Drosophila Notch protein, the Nematode lin-12 and glp-1 proteins, and the closely related vertebrate homologs, Motch (mouse Notch), Xotch (Xenopus Notch), rat Notch, and TAN 1 (human Notch) are membrane bound receptor molecules that control the specification of cell fate for a variety of cell types early in embryogenesis (Rebay et al., 1991, Cell 67:687; Hutter and Schnabel, 1994, Development 120:2051; Del Amo et al 1992, Development 115:737; Reaume et al. 1992 Develop. Biol. 154:377; and Ellisen et al., 1991, Cell 66:649). Specific EGF-like repeats in the Notch receptors are binding sites that attach to protein ligands leading to signal transduction (Rebay et al., 1991 Cell 67:687; Couso and Arias, 1994, Cell 79:259; Fortini and Artavanis-Tsakonas, 1994, Cell 79:273; Henderson et al., 1994, Development 120:2913). Extracellular matrix proteins such as thrombospondin, entactin, tenascin and laminin play key roles in morphogenesis by providing the physical scaffold to which cells attach to form and maintain tissue morphologies (Frazier, 1987, J. Cell. Biol. 105:625; Taraboletti et al., 1990, J. Cell. Biol. 111:765; Ekblom et al., 1994, Development 120:2003).
2.3. DISCOIDIN I/FACTOR VIII-LIKE DOMAINS
A homologous domain structure has been discovered in coagulation factors VIII and V (Kane and Davie, 1986, Proc. Natl. Acad. Sci. U.S.A. 83:6800). This domain is related to a more ancient structure first observed in the discoidin I protein produced by the cellular slime mold Dictyostelium discoideum. Discoidin I is a carbohydrate binding lectin secreted by Dictyostelium cells during the process of cellular aggregation and is involved in cell-substratum attachment and ordered cell migration (Springer et al., 1984, Cell 39:557).
Discoidin I/factor VIII-like domains have also been observed in a number of other proteins. For example, milk fat globule protein (BA46), milk fat globule membrane protein (MFG-E8), breast cell carcinoma discoidin domain receptor (DDR), and the Xenopus neuronal recognition molecule (A5) (Stubbs et al., 1990, Proc. Natl. Acad. Sci. U.S.A. 87:8417; Larocca et al., 1991, Cancer Res. 51:4994; Johnson et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5677). The discoidin I/factor VIII-like domains of the vertebrate proteins are all distantly related to the Dictyostelium sequence but more closely related to each other.
Discoidin I/factor VIII-like domains are rich in positively charged basic amino acids and are believed to bind to negatively charged substrates such as anionic phospholipids or proteoglycans. Both of the milk fat globule proteins have been shown to associate closely with cell membranes and the coagulation factors VIII and V interact with specific platelet membrane proteins (Stubbs et al., 1990 Proc. Nat. Acad. Sci. U.S.A. 87:8417; Larocca et al., 1991, Cancer Res. 51:4994).